In a growing number of fields such as clinical medicine, medical and biological research, homeland security, food safety, and water quality, there is an increasing need for the rapid detection of organisms. In particular, there is a critical need for rapid, field-deployable, and highly sensitive methods, which are more dependable and robust than the commonly used polymerase chain reaction (PCR). Specific nucleotides provide evidence of the presence of microbes (such as bacteria, viruses, and protozoa) in the environment.
To address the need for faster, more sensitive and more accurate detection of biological molecules, nucleotide biosensors have been developed. The majority of biosensors commonly used are optical sensors that measure the fluorescence of molecular markers. The markers quantify the presence of particular nucleotides. These fluorescence-based optical biosensors are highly sensitive, but require complicated reagents and markers, expensive instrumentation, and sophisticated numerical algorithms to interpret the data. These requirements typically limit these methods to use in research laboratories.
Electrochemical nucleotide biosensors have been developed that use measurements connected with the oxidation or reduction of nucleotides. Sensors that utilize direct oxidation of the nucleotide exhibit good sensitivity, but this technique requires the use of high electric potentials, which introduces significant background signals that must be filtered. Other sensors utilize the indirect oxidation of nucleotides using a redox moiety that is bound to certain locations in the nucleotide sequence, such as the guanine bases. However, indirect oxidation sensors have several disadvantages including the difficulty of preparing the probe's substrate, the destruction of the sample during the sensing process, and the dependence on complex sensor instrumentation to ensure high sensitivity.
Other sensor designs use cyclic voltammetric methods coupled with indirect nucleotide oxidation to detect the presence of a target nucleotide. Using cyclic voltammetry methods, these sensors measure the flow of electrons through the sensor electrode in response to an applied time-varying electric potential. The sensors that utilize this design are sensitive, and require less complex instrumentation. However, the substrates typically used to fabricate these sensors are difficult to prepare, due to the complexity of the probe nucleotide molecules used in the sensors. In one design, the probe molecule is a nucleotide attached to a compound that must change conformation after the nucleotide is hybridized in order to generate a signal. In a second design, a signal is generated by the hybridization of the probe nucleotide by both the target nucleotide, and an additional signal nucleotide.
Several other sensor designs increase the probe's available surface area by incorporating nanostructures into the sensor architecture. In this type of sensor design, nanostructures such as carbon nanotubes (CNTs) are affixed to each of two electrical terminals of the sensor in precise alignment, resulting in an electrical connection between the two terminals. Although these designs are more sensitive than previous designs due to their increased surface area, the fabrication of these sensors requires a difficult and expensive process of tightly controlled deposition of CNTs in order to assure that appropriate electrical connections are achieved.
There is a need in the art for a nucleotide detection device that is sensitive, simple to fabricate, does not require complicated measuring equipment, and that can be used outside of the laboratory environment. A need exists for a nucleotide detection device that combines the advantages of cyclic voltammetric measurement techniques, indirect nucleotide oxidation methods, and nanostructures, yet minimizes the disadvantages previously associated with these features. To enhance affordability, the sensor should conduct measurements using relatively inexpensive supplies and equipment, and should be reusable.